JP2001046054A - Alcohol-resistant yeast - Google Patents
Alcohol-resistant yeastInfo
- Publication number
- JP2001046054A JP2001046054A JP21985399A JP21985399A JP2001046054A JP 2001046054 A JP2001046054 A JP 2001046054A JP 21985399 A JP21985399 A JP 21985399A JP 21985399 A JP21985399 A JP 21985399A JP 2001046054 A JP2001046054 A JP 2001046054A
- Authority
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- Japan
- Prior art keywords
- yeast
- agar medium
- sake
- strain
- alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Alcoholic Beverages (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】酒類の製造における酵母に関す
る。特に、従来のアルコール耐性酵母を用いた清酒製造
において問題となっているリンゴ酸などの有機酸の多量
生産が改良された酵母に関する。The present invention relates to yeast in the production of alcoholic beverages. In particular, the present invention relates to a yeast in which the production of a large amount of an organic acid such as malic acid, which is a problem in sake production using a conventional alcohol-resistant yeast, has been improved.
【0002】[0002]
【従来の技術】清酒醪発酵においてアルコール濃度が1
0%を越えると、酵母は自ら生成したアルコールにより
ダメージを受けるものが現れはじめ、アルコール濃度が
15%を越え20%近くに達すると、死滅するものも観
察され、このダメージを受けた酵母および死滅した酵母
から種々の物質が漏出されると考えられている。この漏
出された菌体内物質が製成される清酒に雑味を与えた
り、製成された清酒の品質劣化速度を速くすることか
ら、アルコールに耐性を持つ菌株の探索が盛んに行われ
てきた。例えば、菅野らは、酒造場から野生のアルコー
ル耐性酵母を分離している(J.Brew.Soc.J
apan,65,902(1970))。また、原ら
は、アルコールを含む麹エキス培地でアルコール耐性酵
母を育種、分離し(J. Soc.Brew. Jap
an,71,301(1976))、この系統から日本
醸造協会が頒布している協会11号が誕生した。2. Description of the Related Art Alcohol concentration is 1 in sake mash fermentation.
If it exceeds 0%, some yeasts begin to be damaged by the alcohol generated by themselves, and when the alcohol concentration exceeds 15% and approaches 20%, some of the yeasts are observed to be killed. It is thought that various substances are leaked from the yeast. The leaked intracellular substances impart a taste to the sake produced and increase the quality deterioration rate of the produced sake, so that strains resistant to alcohol have been actively searched for. . For example, Sugano et al. Have isolated wild alcohol-resistant yeast from a brewery (J. Brew. Soc. J.).
apan, 65 , 902 (1970)). Hara et al. Bred and isolated alcohol-resistant yeast in a koji extract medium containing alcohol (J. Soc. Brew. Japan).
an, 71 , 301 (1976)), and from this strain, Association No. 11 distributed by the Japan Brewing Association was born.
【0003】また、菌体内物質の漏出に関する基礎的な
研究も進められてきた。後藤と土肥は、米および麹には
検出される量は含まれていないが酵母の液胞に多量に蓄
積されるS−アデノシルメチオニン(以下、SAMと略
す。)に注目し、SAMの菌体外への漏出が清酒醪発酵
における酵母菌体内物質漏出の指標となりうることを報
告している(J.Brew.Soc.Japan,8
7,230(1992))。また、アルコール耐性酵母
を用いた清酒醪発酵では、SAMの漏出が少ないことが
明らかにされている。[0003] Basic research on leakage of intracellular substances has also been advanced. Goto and Toi paid attention to S-adenosylmethionine (hereinafter abbreviated as SAM), which is not contained in rice and koji but contained in a large amount in the vacuoles of yeast, and detected bacteria of SAM. It has been reported that extracorporeal leakage can be an indicator of yeast intracellular substance leakage in sake mash fermentation (J. Brew. Soc. Japan, 8).
7 , 230 (1992)). In addition, it has been revealed that in the sake mash fermentation using alcohol-resistant yeast, the leakage of SAM is small.
【0004】[0004]
【本発明が解決しようとする課題】現在、清酒製造に最
も頻繁に用いられているアルコール耐性菌株は協会11
号であり、他の菌株は殆ど用いられていない。しかも、
協会11号により製成される清酒は、協会7号や協会9
号、協会701号などの一般に広く使われている酵母菌
株により製成される清酒と比較するとリンゴ酸を非常に
多く含み、一般の清酒と較べるとやや異なった香味であ
るなどの難点があり、協会11号が清酒製造に用いられ
るのは限られた場合にしかすぎない。At present, the most frequently used alcohol-resistant strain for sake production is the Association 11
And other strains are rarely used. Moreover,
Sake produced by Association No. 11 is Association No. 7 and Association No. 9
No., compared with sake produced by commonly used yeast strains such as the Association No. 701, contains much more malic acid, and has a disadvantage that it has a slightly different flavor as compared to ordinary sake. The Association No. 11 is used for sake production only in limited cases.
【0005】原らは、前記のアルコール耐性酵母を育
種、分離とは別に、菌体溶解酵素を用いたアルコール耐
性酵母の育種、分離を行っているが、この結果において
もアルコール耐性の強い菌株にはリンゴ酸高生産が認め
られている(J.Brew.Soc.Japan,7
3,408(1978)。そのため、菌体内物質の漏出
が少ないというアルコール耐性酵母の利点を保持しなが
ら一般の清酒の範疇の香味を有する清酒を製成する新た
な性質を示すアルコール耐性酵母の出現が望まれ、より
広い目的でのアルコール耐性酵母の使用が望まれてい
る。Hara et al. Breed and isolate alcohol-tolerant yeast using a cell lysing enzyme, apart from breeding and isolating the above-mentioned alcohol-tolerant yeast. Shows high production of malic acid (J. Brew. Soc. Japan, 7
3 , 408 (1978). Therefore, the emergence of alcohol-tolerant yeast exhibiting new properties for producing sake having a flavor in the category of general sake while retaining the advantage of alcohol-tolerant yeast that the leakage of intracellular substances is small is desired. It is desired to use alcohol-resistant yeasts in yeasts.
【0006】[0006]
【課題を解決するための手段】上記課題を解決するため
に、清酒醪発酵が完了した後も長期間にわたり静置を続
けて高濃度アルコールが存在する条件下でアルコール耐
性株を育種し、このなかから様々な方法で優れた性質を
有するアルコール耐性株を探索した。様々な探索を試み
ていたところ、醪発酵においてSAM漏出が少ないとい
う基準で選択した菌株のなかに、YNBG寒天培地(デ
ィフコ社製イースト・ナイトロジェン・ベース0.67
%、グルコース2%、寒天2%)から麹エキス寒天培地
に継代した場合は良好に生育するが、YPD寒天培地
(酵母エキス1%、ペプトン2%、グルコース2%、寒
天2%)に継代した場合やYNBG寒天培地に継代した
場合では生育が不良な菌株が存在することを見出した。
さらに、これらの菌株が製成する清酒について調べたと
ころ、リンゴ酸などの有機酸生成に問題のない良好な清
酒を製成する菌株が多く存在することを見出し、本発明
を完成させた。Means for Solving the Problems In order to solve the above-mentioned problems, alcohol-resistant strains are bred under the condition that high-concentration alcohol is present by continuing to stand for a long time even after completion of sake mash fermentation. Among them, various methods were used to search for alcohol-resistant strains having excellent properties. While trying various searches, among the strains selected on the basis of low SAM leakage in mash fermentation, YNBG agar medium (Difco's yeast nitrogen base 0.67) was selected.
%, Glucose 2%, and agar 2%), it grows well when subcultured on a koji extract agar medium, but it is grown on a YPD agar medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar). It was found that some strains with poor growth existed when the culture was subcultured or subcultured on a YNBG agar medium.
Furthermore, when the sake produced by these strains was examined, it was found that there were many strains producing good sake which had no problem in producing organic acids such as malic acid, and completed the present invention.
【0007】すなわち、本発明は、YNBG培地に継代
後YPD培地及びYNBG培地に継代すると生育が不良
であるという新しいタイプのアルコール耐性酵母菌株を
提供するものであり、本発明の菌株は、アルコール耐性
株でありながら、リンゴ酸などの有機酸含量が一般の清
酒と同等である良好な清酒を製成することができるもの
である。[0007] That is, the present invention provides a new type of alcohol-resistant yeast strain that grows poorly when it is subcultured into a YPD medium or YNBG medium after being subcultured into a YNBG medium. Although it is an alcohol-resistant strain, it is possible to produce good sake having an organic acid content such as malic acid equivalent to that of general sake.
【0008】本発明の酵母は、醪発酵が完了した後も長
期間にわたり静置を続けたものから分離した酵母菌株か
ら探索することにより取得することができる。ここで、
醪の調製に用いる酵母は、協会7号、9号または701
号など通常の清酒製造に用いられているサッカロミセス
・セレビシエに属する酵母菌株をはじめ様々な酵母を用
いることができ、また、酵母を変異剤、紫外線、放射線
などで変異処理したものを用いても良いが、変異処理を
せずに自然誘発で起きる変異により目的の菌株を得るこ
とができる。[0008] The yeast of the present invention can be obtained by searching for yeast strains separated from those that have been left standing for a long period of time even after the completion of the mash fermentation. here,
The yeast used for mash preparation is Kyokai No. 7, No. 9 or 701.
Various yeasts can be used, including yeast strains belonging to Saccharomyces cerevisiae, which are used in ordinary sake production such as No. 1, and yeasts that have been mutated with a mutagen, ultraviolet rays, radiation, etc. may be used. However, a desired strain can be obtained by a mutation caused by spontaneous induction without performing a mutation treatment.
【0009】醪の調製は、通常の清酒製造に用いられて
いる米、麹及び水を用いればよく、必要に応じて乳酸や
酵素剤などを加えてもよい。また、醪の調製方法は、従
来の醪発酵の方法を適宜用いればよく、好ましくは、1
0〜20℃の温度で留後8〜20日にアルコール濃度が
18%を越える発酵条件で行うのがよい。また、発酵が
完了した醪の静置は、10〜20℃の温度で30日以上
続けるのが好ましく、長期間静置した後の醪の酵母を用
いて再度醪発酵を行い、再度長期間静置してもよい。For the preparation of the mash, rice, koji and water used in ordinary sake production may be used, and if necessary, lactic acid or an enzyme agent may be added. In addition, the method of preparing the mash may be a conventional mash fermentation method as appropriate, and is preferably 1
The fermentation is preferably carried out 8 to 20 days after the distillation at a temperature of 0 to 20 ° C. under fermentation conditions in which the alcohol concentration exceeds 18%. Moreover, it is preferable that the fermentation of the fermented mash is continued at a temperature of 10 to 20 ° C. for 30 days or more. May be placed.
【0010】発酵が完了した後も長期間にわたり静置を
続けた醪からの酵母菌株の分離は、一般の方法を用いれ
ばよいが、好ましくは、醪を生理食塩水で希釈したもの
を麹エキス寒天培地に塗布し、15〜30℃で静置培養
してコロニーを形成させ、コロニーから麹エキス寒天培
地のスラントに植え継ぐのがよい。The yeast strain can be separated from the mash which has been kept still for a long period of time even after the fermentation is completed, using a general method. Preferably, the mash is diluted with physiological saline and the koji extract is used. It is preferable to apply the solution to an agar medium, and to incubate the culture at 15 to 30 ° C. statically to form a colony. Then, the colony is preferably transferred to a slant of a koji extract agar medium.
【0011】分離した菌株のなかからSAM漏出が少な
い菌株を選択するには、各分離菌株及び親株を用いて醪
発酵の小仕込を行い、それぞれの醪上清のSAM濃度と
アルコール濃度の経時変化を測定し、アルコール濃度が
18%に到達したときの醪上清SAM濃度を親株のもの
と比較してSAM漏出が少ないかを判定することにより
行うことができる。このとき、SAM濃度の測定は、た
とえば、後藤と土肥の方法(J.Brew.Soc.J
apan,87,230(1992))で行うことがで
き、また、アルコール濃度の測定は、たとえば、国税庁
所定分析法注解に記載の方法に従って行うことができ
る。In order to select a strain having a small amount of SAM leakage from the isolated strains, a small preparation of mash fermentation is performed using each of the isolated strains and the parent strain, and the SAM concentration and alcohol concentration of each mash supernatant are changed over time. And the SAM concentration of the mash supernatant when the alcohol concentration reaches 18% is compared with that of the parent strain to determine whether SAM leakage is small. At this time, the SAM concentration is measured, for example, by the method of Goto and Toi (J. Brew. Soc. J.
ap., 87 , 230 (1992)), and the measurement of the alcohol concentration can be performed, for example, according to the method described in the comment of the prescribed analysis method of the NTA.
【0012】次に、上記で得られたSAM漏出が少ない
菌株の中から、YPD寒天培地及びYNBG寒天培地で
は生育が不良である菌株の選択するには、上記のSAM
漏出が少ないと判定された菌株をまずYNBG寒天培地
のスラントに植え継ぎ、次にこのスラントから麹エキス
寒天培地、YPD寒天培地及びYNBG寒天培地に各々
移植し、この3種の寒天培地を30℃で3日間静置し
て、目視により生育状態を判定することにより、麹エキ
ス寒天培地では生育が良好であるがYPD寒天培地及び
YNBG寒天培地では生育が不良なものを取得すること
ができる。ここで言う麹エキス培地とは、麹米に2倍重
量の水を加え、50℃で1晩静置して麹菌の酵素により
米の糖化を進めたものをろ過して得た液体から調製した
培地である。麹米とは、麹室で、蒸した米を40℃付近
まで放冷したところに麹菌を振りかけて、米に麹菌を増
殖させたものであり、米の澱粉を糖化する酵素が含まれ
ている。また、生育の良、不良の判断は、接種面一面に
菌体が増産する場合は良好、接種面に点々と小さな菌体
の塊のみしか形成されない場合は不良と判断すればよ
い。Next, from among the strains with low SAM leakage obtained above, those strains that do not grow well on the YPD agar medium and the YNBG agar medium are selected from the SAMs described above.
The strain determined to have little leakage was first transferred to a slant of a YNBG agar medium, and then transplanted from the slant to a koji extract agar medium, a YPD agar medium and a YNBG agar medium, and the three kinds of agar medium were heated at 30 ° C. For 3 days, and visually determine the growth state, whereby it is possible to obtain a koji extract agar medium that has good growth but a YPD agar medium and a YNBG agar medium that have poor growth. The koji extract medium mentioned here was prepared from a liquid obtained by adding twice the weight of water to koji rice, allowing the koji rice to stand at 50 ° C. overnight, and saccharifying the rice with the enzyme of the koji mold, followed by filtration. It is a medium. Koji rice is a steamed rice that is allowed to cool to around 40 ° C in a koji room and sprinkled with koji mold to grow the koji mold on the rice, and contains an enzyme that saccharifies rice starch. . In addition, good or bad growth can be judged as good when the bacterial cells increase over the whole inoculated surface, and bad when only small and small lump of bacterial cells are formed on the inoculated surface.
【0013】このようにして、醪発酵においてSAM漏
出が少なく、かつ、YNBG寒天培地に継代するとYP
D寒天培地及びYNBG寒天培地では生育が不良である
性質を持つ酵母菌株を取得することができる。このよう
にして得られた本発明の菌株は、通常の清酒醪発酵にお
いてアルコール濃度が18%に到達したときの醪上清S
AM濃度が協会7号の50%以下であり、また、アルコ
ール濃度が18%に到達したときのメチレンブルー染色
による酵母の死滅率は10%以下であった。[0013] Thus, when the SAM fermentation is low in the mash fermentation and the YNBG agar medium is subcultured, the YP
A yeast strain having poor growth properties can be obtained on the D agar medium and the YNBG agar medium. The bacterial strain of the present invention thus obtained is a mash supernatant S when the alcohol concentration reaches 18% in normal sake mash fermentation.
The AM concentration was 50% or less of Association No. 7, and the killing rate of yeast by methylene blue staining when the alcohol concentration reached 18% was 10% or less.
【0014】本発明の菌株は、YPD寒天培地及びYN
BG寒天培地で生育が不良である他、糖蜜培地(糖蜜1
0%、硫酸アンモニウム0.5%、尿素1%、リン酸二
水素一カリウム0.5%、硫酸マグネシウム0.05
%)を用いた通気撹拌培養での菌体の収量も協会7号の
場合と較べると20〜30%にすぎなかった。これらの
ことから、具体的には物質を特定できないが麹エキスに
含まれるものを本発明の菌株は要求すると考えられる。
アルコール耐性酵母のなかでこのような栄養要求性を持
つものは、これまでに知られておらず、本発明の菌株は
新規タイプのアルコール耐性酵母であることが判明し
た。そこで、取得した菌株の内の1株をSAM001と
命名し、本株を受託番号FERM P−17373とし
て通商産業省工業技術院生命工学工業技術研究所に寄託
した。The strain of the present invention comprises YPD agar medium and YN
In addition to poor growth on BG agar medium, molasses medium (molasses 1
0%, ammonium sulfate 0.5%, urea 1%, monopotassium dihydrogen phosphate 0.5%, magnesium sulfate 0.05
%), The yield of bacterial cells in aeration and stirring culture was only 20 to 30% as compared with the case of Association No. 7. From these facts, it is considered that the strain of the present invention requires what is contained in the koji extract, although the substance cannot be specifically identified.
No alcohol-tolerant yeast having such auxotrophy has been known so far, and it has been found that the strain of the present invention is a novel type of alcohol-tolerant yeast. Therefore, one of the obtained bacterial strains was named SAM001, and this strain was deposited as Accession No. FERM P-17373 with the Biotechnology Industrial Research Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry.
【0015】SAM001株は、協会7号を用いて前記
の方法により得られた菌株であり、TTC染色およびβ
−アラニン培地での繁殖性は親株である協会7号と同じ
であった。SAM001株のアルコール耐性能力の強さ
を調べるために、本株の親株である協会7号及びアルコ
ール耐性株のなかで清酒製造に最も用いられている協会
11号と比較して、16%アルコール存在下での生存能
力、10%アルコール存在下での増殖能力および18%
アルコール存在醪での発酵能力を測定した。その結果を
表1に示す通り、SAM001株のアルコール耐性能力
は協会11号とは異なることが明らかになった。The SAM001 strain is a strain obtained by the above-mentioned method using Kyokai No. 7;
-Fertility in alanine medium was the same as that of the parent strain, Association No. 7. In order to examine the strength of the SAM001 strain in alcohol tolerance, 16% alcohol was present in comparison to the parent strain of the present strain, Association No. 7 and the alcohol-resistant strain, Association No. 11, which is most used for sake production. Viability in the presence of, 10% growth in the presence of alcohol and 18%
The fermentation ability of alcoholic mash was measured. As shown in the results in Table 1, it was revealed that the alcohol tolerance ability of the SAM001 strain was different from that of the Association No. 11.
【0016】[0016]
【表1】 [Table 1]
【0017】また、本発明には、本発明の酵母を用いて
製造した酒類も含まれる。本発明の酵母は、清酒をはじ
め焼酎、ビール、ワインなど様々な酒類の製造に用いる
ことが出来き、また、本発明の菌株を用いた酒類の製成
は、従来の酒類の製造方法を適宜用いることにより可能
である。The present invention also includes alcoholic beverages produced using the yeast of the present invention. The yeast of the present invention can be used for the production of various alcoholic beverages such as sake, shochu, beer, wine, and the like, and the production of alcoholic beverages using the bacterial strain of the present invention is performed by appropriately employing the conventional alcoholic beverage production method. It is possible by using.
【0018】アルコール耐性株のなかで清酒製造に最も
用いられている協会11号により製成される清酒は、協
会7号や協会9号、協会701号などの通常の清酒酵母
により製成した清酒と比較するとリンゴ酸などが多量に
含まれており有機酸組成が異なることが知られている
が、本発明の菌株の製成する清酒について有機酸分析を
行ったところ、リンゴ酸をはじめ種々の有機酸含量が親
株(協会7号)のものと同等である菌株が多く存在し
た。Among the alcohol-tolerant strains, sake produced by Association No. 11 which is most used for sake production is sake produced by ordinary sake yeast such as Association No. 7, Association No. 9 and Association No. 701. It is known that malic acid and the like are contained in a large amount and the organic acid composition is different as compared with the above.However, when an organic acid analysis was performed on sake produced by the strain of the present invention, various acids including malic acid were There were many strains whose organic acid content was equivalent to that of the parent strain (Kyoto 7).
【0019】また、製成した清酒の清酒らしさを官能評
価により判定したところ、総ての菌株のものが協会11
号により製成されたものより清酒らしさがあった。ま
た、官能評価により香味の良さを判定したところ、親株
のものよりも優れているものが多く存在した。このこと
から、従来の清酒の香味を持ち、良好な酒質の清酒を製
成することが可能な菌株が多く含まれると推測された。
以下、具体例を挙げて本発明を説明するが、本発明は以
下の具体例に限定されるものではない。The quality of the produced sake was determined by sensory evaluation.
There was more sake taste than the one made by No. In addition, when good flavor was determined by sensory evaluation, there were many that were superior to those of the parent strain. From this, it was presumed that a large number of bacterial strains having the flavor of conventional sake and capable of producing sake with good sake quality were included.
Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited to the following specific examples.
【0020】[0020]
【実施例1】協会7号酵母を10mlYPD培地に種菌
し、30℃で1日間静置培養した。この培養液を400
ml糖蜜培地に移植し、30℃で3日間静置培養した。
この培養液を集菌洗浄し、この酵母菌体を用い、表2に
示す仕込配合で清酒醪発酵を行った。Example 1 Yeast No. 7 was inoculated into 10 ml YPD medium and cultured at 30 ° C. for 1 day. This culture was added to 400
The cells were transplanted into a ml molasses medium and cultured at 30 ° C. for 3 days.
The culture solution was collected and washed, and the sake mash fermentation was performed using the yeast cells and the blended ingredients shown in Table 2.
【0021】[0021]
【表2】 [Table 2]
【0022】ここで、原料である米は日本晴(平成7年
度産)を精米歩合70%に精米して使用した。また、麹
は常法により2日間麹室にて製麹し、酵素剤はスピター
ゼMKを用いた。仕込み方法は3日間に分け、初日を初
添、1日置いて3日目に仲添、4日目に留添をし、15
℃で発酵させた。酵母は初添時に湿菌体重量で1g添加
した。The rice used as the raw material was Nipponbare (produced in 1995), which was polished to a polishing rate of 70%. In addition, koji was made in a koji room for 2 days by a conventional method, and spitase MK was used as an enzyme preparation. The preparation method was divided into 3 days, the first day was the first soup, the first day was left, the third day was the middle soup and the fourth day was the soup.
Fermented at 0 ° C. At the time of the initial addition, 1 g of yeast was added by wet cell weight.
【0023】留後17日に醪上清のアルコール濃度が1
9.3%に達した。その後も15℃で静置を続け、留後
60日の醪をサンプリングし、無菌生理食塩水で希釈し
た液を麹エキス寒天培地に塗布した。この寒天培地を3
0℃で4日間静置培養し、コロニーを形成させ、各コロ
ニーからアルコール耐性菌株探索の候補菌株のスラント
を調製した。On the 17th day after the distillation, the alcohol concentration of the mash supernatant was 1
Reached 9.3%. Thereafter, the mixture was allowed to stand still at 15 ° C., the moromi was sampled 60 days after the distillation, and a liquid diluted with sterile physiological saline was applied to the koji extract agar medium. Add this agar medium to 3
The culture was allowed to stand still at 0 ° C. for 4 days to form colonies, and a slant of a candidate strain for searching for an alcohol-resistant strain was prepared from each colony.
【0024】ここで得られた株の内の1株である本発明
の菌株SAM001株、及び親株である協会7号を用い
て上記の方法で培養し、集菌洗浄した酵母菌体を用いて
表2に示す仕込配合で清酒醪発酵を行った。このときの
醪上清のアルコール濃度およびSAM濃度を測定し、そ
の結果をそれぞれ表3および表4に示す。Using the yeast SAM001, which was cultured by the above method using the SAM001 strain of the present invention, which is one of the obtained strains, and the parent strain, Association No. 7, was collected and washed. Sake mash fermentation was carried out with the blending composition shown in Table 2. At this time, the alcohol concentration and the SAM concentration of the mash supernatant were measured, and the results are shown in Tables 3 and 4, respectively.
【0025】[0025]
【表3】 [Table 3]
【0026】[0026]
【表4】 [Table 4]
【0027】表3に示す通り、本発明の菌株のアルコー
ル生成速度は、醪発酵前半では親株よりもやや遅かった
が醪発酵後半では親株よりも速く、アルコール生成量は
醪発酵末期に親株のアルコール生成量を追い越した。ま
た、表4に示す通り、本発明の菌株のSAM漏出量は親
株と比較すると著しく少なく、アルコール濃度が18%
に到達したときのSAM濃度を基にSAM漏出量を比較
したところ、本発明の菌株のSAM漏出量は親株の3
1.6%であった。As shown in Table 3, the alcohol production rate of the strain of the present invention was slightly slower in the first half of the mash fermentation than in the parent strain, but was faster in the second half of the mash fermentation than in the parent strain. Overtake production. Further, as shown in Table 4, the amount of SAM leakage of the strain of the present invention was significantly smaller than that of the parent strain, and the alcohol concentration was 18%.
When the amount of SAM leaked was compared based on the SAM concentration at the time when the strain reached, the amount of SAM leaked from the strain of the present invention was 3% of that of the parent strain.
1.6%.
【0028】ここで、SAM濃度の測定は、後藤と土肥
の高速液体クロマトグラフィーによる方法(J.Bre
w.Soc.Japan,87,230(1992))
に準じて行った。すなわち、強カチオン交換樹脂カラム
Chemocopak Nucleosil 10SA
を用い、リン酸アンモニウム緩衝液(pH3.0)の
0.05Mから0.5Mのグラジエントで展開し、26
0nmの吸光度で検出することにより行った。Here, the SAM concentration was measured by a method using high performance liquid chromatography of Goto and Toi (J. Bre
w. Soc. Japan, 87 , 230 (1992))
It went according to. That is, a strong cation exchange resin column Chemopakak Nucleosil 10SA
And developed with a gradient of 0.05 M to 0.5 M of ammonium phosphate buffer (pH 3.0) using 26
The detection was performed by detecting the absorbance at 0 nm.
【0029】[0029]
【実施例2】本発明の菌株SAM001株および親株で
ある協会7号をYNBG寒天培地に植え、30℃で4日
間静置培養してスラントを作製した。このスラントから
麹エキス寒天培地及びYPD寒天培地並びにYNBG寒
天培地に移植し、30℃で静置培養し、3日後に目視に
より生育状況を判定した。その結果を表5に示す。Example 2 The strain SAM001 of the present invention and the parent strain Kyokai No. 7 were inoculated on a YNBG agar medium and allowed to stand at 30 ° C. for 4 days to produce a slant. The slant was transplanted to a koji extract agar medium, a YPD agar medium, and a YNBG agar medium, cultured at 30 ° C. statically, and three days later, the growth status was visually determined. Table 5 shows the results.
【0030】[0030]
【表5】 [Table 5]
【0031】表5に示す通り、親株である協会7号は総
ての培地で生育良好であった。本発明の菌株は、麹エキ
ス寒天培地では生育は良好であったが、YPD寒天培地
およびYNBG寒天培地では親株である協会7号のもの
と比較すると寒天表面に形成される菌体の塊の厚みが薄
く生育不良であると判定した。特に、YNBG寒天培地
の生育においては、親株のものは、菌を接種した部分に
は一面に菌体が増殖しているのに対して、本発明の菌株
のものは、点々と小さな菌体の塊があるのみであり、明
らかに生育不良であることが観察された。As shown in Table 5, the parent strain, Association No. 7, grew well in all media. Although the strain of the present invention grew well on the koji extract agar medium, the thickness of the cell mass formed on the agar surface was higher on the YPD agar medium and the YNBG agar medium than on the parent strain, Association No. 7. Was thin and the growth was poor. In particular, in the growth of the YNBG agar medium, the cells of the parent strain grow on the entire surface of the portion inoculated with the bacteria, whereas the cells of the strain of the present invention show a small number of small cells. It was observed that there were only lumps and clearly poor growth.
【0032】[0032]
【実施例3】本発明の菌株、親株及び協会11号を麹エ
キス培地に種菌し、30℃で1日間静置培養した。これ
らの培養液にアルコール濃度が16%となるようにアル
コールを加え、30℃に4時間温置し、メチレンブルー
染色で死滅率を測定した。この結果を表6に示す。Example 3 The strain, parent strain and Kyokai No. 11 of the present invention were inoculated in a koji extract medium and cultured at 30 ° C. for 1 day. Alcohol was added to these cultures so that the alcohol concentration became 16%, incubated at 30 ° C. for 4 hours, and the mortality was measured by methylene blue staining. Table 6 shows the results.
【0033】[0033]
【表6】 [Table 6]
【0034】表6に示す通り、本発明の菌株の高アルコ
ール濃度中での生存維持能力は、協会11号ほどは強く
なかったが、親株よりは強かった。As shown in Table 6, the ability of the strain of the present invention to maintain viability at a high alcohol concentration was not as strong as Association No. 11, but was stronger than the parent strain.
【0035】[0035]
【実施例4】本発明の菌株、親株及び協会11号を麹エ
キス培地に種菌し、30℃で1日間静置培養した。これ
らの培養液を10%アルコール含有麹エキス培地に酵母
密度が約104/mlとなるように移植し、30℃で2
0時間静置培養し、トーマ血球板を用いて生菌数を測定
して増殖倍率を求めた。この結果を表7に示す。EXAMPLE 4 The strain, parent strain and No. 11 of the present invention were inoculated in a koji extract medium and cultured at 30 ° C. for 1 day. These cultures were transplanted to a 10% alcohol-containing koji extract medium at a yeast density of about 10 4 / ml.
The culture was allowed to stand still for 0 hour, and the number of viable cells was measured using a toma blood cell plate to determine the growth rate. Table 7 shows the results.
【0036】[0036]
【表7】 [Table 7]
【0037】表7に示す通り、本発明の菌株の高アルコ
ール濃度中での増殖速度は、親株および協会11号より
も速かった。As shown in Table 7, the growth rate of the strain of the present invention at a high alcohol concentration was faster than that of the parent strain and Kyokai No.11.
【0038】[0038]
【実施例5】本発明の菌株を麹エキス培地10mlに4
本種菌し、30℃で1日間静置培養した。この培養液1
本分を麹エキス培地500mlに移植し、30℃で2日
間静置培養し、合計2Lの培地に増殖させた本発明の菌
株の菌液を調製した。この菌液を水100Lおよび乳酸
1Lを入れた容器に加えた後に、麹米45kg、次いで
70%精白米105kgを加え、常法により速醸酒母を
調製した。この調製した酒母を用いて、表8に示す仕込
配合で醸造を行った。Example 5 A strain of the present invention was added to 10 ml of a koji extract medium in 4 ml.
The inoculum was cultured at 30 ° C. for 1 day. This culture solution 1
This was transplanted to a koji extract medium (500 ml) and cultured at 30 ° C. for 2 days to prepare a bacterial solution of the strain of the present invention grown in a total of 2 L of medium. After adding this bacterial solution to a container containing 100 L of water and 1 L of lactic acid, 45 kg of koji rice and then 105 kg of 70% polished rice were added, and a quick-brewed sake mother was prepared by a conventional method. Using this prepared sake mother, brewing was carried out with the blended composition shown in Table 8.
【0039】[0039]
【表8】 [Table 8]
【0040】ここで、原料である米は日本晴(平成10
年度産)を精米歩合70%に精米して使用し、、麹は常
法により2日間麹室にて製麹した。仕込み方法は3日間
に分け、初日を初添、1日置いて3日目に仲添、4日目
に留添をし、15℃で発酵させた。酒母は、調製した全
量を初添時に加えた。Here, rice as a raw material is Nipponbare (Heisei 10
The koji was produced in a koji room for 2 days by a conventional method. The preparation method was divided into 3 days, the first day was the first addition, the first day was left, the middle day was added on the third day, the fourth day was added, and the fermentation was carried out at 15 ° C. The sake mother added the whole amount prepared at the time of the first addition.
【0041】また、留19日に30%アルコールを87
6L添加し、留20日に米120Kgを汲み水240L
のなかで酵素剤スピターゼMを用いて55℃で12時間
の糖化をして15℃まで冷却したものを四段として加え
た。留21日に上槽し、アルコール濃度20.7%、日本酒
度+7.5,酸度1.7、アミノ酸度1.7の製成酒5681
Lを得た。On the 19th day, 30% alcohol was added to 87%.
Add 6 L, pump 120 kg of rice on the 20th and pump 240 L of water
Among them, saccharification was performed at 55 ° C for 12 hours using an enzyme agent spitase M, and the mixture was cooled to 15 ° C and added in four stages. On the 21st day of the distillery, the upper tank was made, and the alcohol concentration was 20.7%, sake degree +7.5, acid degree 1.7, and amino acid degree 1.7.
L was obtained.
【0042】親株を用いて同様な方法で醸造した製成酒
は、アルコール濃度20.4%、日本酒度+5.0 ,
酸度1.8、アミノ酸度1.5であり、日本酒度を比較
すると本発明の菌株から製成した清酒は高く、本発明の
菌株を用いて辛口タイプの清酒を製成できることが明ら
かになった。The sake brewed in the same manner using the parent strain had an alcohol concentration of 20.4%, a sake degree of +5.0,
The acidity was 1.8 and the amino acid content was 1.5. Compared with the sake degree, sake produced from the strain of the present invention was high, and it was revealed that dry type sake could be produced using the strain of the present invention. .
【0043】また、本製成酒のグルコースおよびマルト
ース濃度を測定したところ、それぞれ1.82%および
0.30%であった。親株を用いて同様な方法で醸造し
た清酒の2.26%および0.45%と較べると、本発
明の菌株により製成される清酒の残存グルコースおよび
マルトースは少なく、辛口タイプの清酒を製成するのに
適していると考えられる。The glucose and maltose concentrations of the present sake were 1.82% and 0.30%, respectively. Compared to 2.26% and 0.45% of sake brewed by the same method using the parent strain, the sake produced by the strain of the present invention has less residual glucose and maltose, and produces dry-type sake. It is considered suitable for
【0044】また、上槽直前の醪中の酵母の死滅率をメ
チレンブルー染色法により観察したところ、19.2%
であり、親株を用いて同様な方法で醸造した場合の5
8.3%と比較するとかなり低かった。また、アルコー
ル濃度が18%に達したときの死滅率は、1.4%であ
り、親株を用いて同様な方法で醸造した場合の15.0
%と比較するとかなり低かった。When the kill rate of yeast in the mash immediately before the upper tank was observed by methylene blue staining, 19.2%
5 when brewed by the same method using the parent strain.
It was much lower than 8.3%. The kill rate when the alcohol concentration reached 18% was 1.4%, which was 15.0 in the case of brewing in the same manner using the parent strain.
It was considerably lower than the percentage.
【0045】アルコール濃度が18%に到達したときの
醪上清SAM濃度は、4.6ppmであり、親株を用い
て同様な方法で醸造した場合の12.6ppmと比較す
るとかなり低かった。本製成酒並びに親株及び協会11
号を用いて本製成酒と同様な方法で醸造した清酒の有機
酸分析を行ったところ、表9に示す結果が得られた。The SAM concentration of the mash supernatant when the alcohol concentration reached 18% was 4.6 ppm, which was considerably lower than 12.6 ppm when brewed by the same method using the parent strain. Honsei Sake, Parent Stock and Association 11
The organic acid analysis of the sake brewed in the same manner as in the present brewed sake using No. No. 10 gave the results shown in Table 9.
【0046】[0046]
【表9】 [Table 9]
【0047】表9の結果から、協会11号により製成さ
れる清酒には協会7号のものと較べるとリンゴ酸および
コハク酸が非常に多量に含まれるが、本発明の菌株によ
り製成される清酒の有機酸組成は、親株である協会7号
のものとあまり変わらないことが確認された。From the results shown in Table 9, the sake produced by the Association No. 11 contains a much larger amount of malic acid and succinic acid than that of the Association No. 7, but it is produced by the strain of the present invention. It was confirmed that the organic acid composition of the sake did not differ much from that of the parent strain, Association No. 7.
【0048】[0048]
【実施例6】実施例5に記載の本発明の菌株、協会7号
及び協会11号により製成された清酒を用いて、15名
のパネルが清酒らしい香味を有しているかを官能評価し
た。評価方法は、協会7号により製成された清酒を基準
とし、5:協会7号により製成された清酒よりも非常に
清酒らしい、4:協会7号により製成された清酒よりも
やや清酒らしい、3:協会7号により製成された清酒と
同等、2:協会7号により製成された清酒と較べるとや
や清酒らしくない、1:協会7号により製成された清酒
と較べると全く清酒らしくない、の5段階評価で行い、
表10に示す結果が得られた。Example 6 Using the strain of the present invention described in Example 5, sake produced by Kyokai No. 7 and Kyokai No. 11, a panel of 15 panelists was subjected to a sensory evaluation to determine whether or not a panel of 15 persons had a flavor like sake. . The evaluation method is based on the sake produced by the Association No.7. 5: The sake is much more refined than the sake produced by the Association No.7. 4: The sake is slightly more refined than the sake produced by the Association No.7. Probable 3: Equivalent to sake made by Association No. 2: Slightly less like sake compared to sake made by Association No. 7, 1: Completely compared to sake made by Association No. 7 Performed on a five-point scale, not like sake.
The results shown in Table 10 were obtained.
【0049】[0049]
【表10】 [Table 10]
【0050】表10に示す通り、本発明の菌株から製成
された清酒は、協会11号により製成された清酒よりも
清酒らしい香味を有していた。As shown in Table 10, the sake produced from the strain of the present invention had a flavor more like sake than the sake produced by Association No. 11.
【0051】[0051]
【実施例7】実施例5に記載の本発明の菌株、協会7号
および協会11号により製成された清酒を用いて、15
名のパネルが良質の香味を有しているかを官能評価し
た。評価方法は、5:非常に良い、4:やや良い、3:
普通、2:やや悪い、1:非常に悪い、の5段階評価で
行い、表11に示す結果が得られた。Example 7 Using the strain of the present invention described in Example 5, the sake produced by Kyokai No. 7 and Kyokai No. 11,
A sensory evaluation was performed to determine whether the name panel had a good quality flavor. The evaluation method is 5: very good, 4: somewhat good, 3:
Normally, the evaluation was performed on a five-point scale of 2: somewhat poor, 1: very poor, and the results shown in Table 11 were obtained.
【0052】[0052]
【表11】 [Table 11]
【0053】表11に示す通り、本発明の菌株から製成
された清酒は、協会7号により製成された清酒よりもや
や香味が良く、協会11号により製成された清酒よりも
良質の香味を有していた。As shown in Table 11, the sake produced from the strain of the present invention has a slightly better flavor than the sake produced by the Association No. 7, and has a higher quality than the sake produced by the Association No. 11. Had a flavor.
Claims (4)
に継代すると良好に生育するが、YNBG寒天培地から
YPD寒天培地に継代してもYNBG寒天培地に継代し
ても生育が不良であるアルコール耐性酵母。Claims 1. Although growth is good when subcultured from YNBG agar medium to koji extract agar medium, growth is poor even when subcultured from YNBG agar medium to YPD agar medium or YNBG agar medium. Alcohol-resistant yeast.
酸の生成量が、協会7号を用いて同条件で清酒製造を行
う場合と比較して同等であることを特徴とする請求項1
に記載の酵母。2. The production amount of malic acid and succinic acid in sake production is equal to that in a case where sake production is performed under the same conditions using Association No. 7 under the same conditions.
The yeast according to 1.
株(FERM P−17373)。3. The yeast according to claim 2, which is SAM001.
Strain (FERM P-17373).
を用いて製造した酒類。4. Alcoholic beverages produced using the yeast strains according to claim 1, 2 and 3.
Priority Applications (1)
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---|---|---|---|
JP21985399A JP4402207B2 (en) | 1999-08-03 | 1999-08-03 | Alcohol-resistant yeast |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP21985399A JP4402207B2 (en) | 1999-08-03 | 1999-08-03 | Alcohol-resistant yeast |
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Publication Number | Publication Date |
---|---|
JP2001046054A true JP2001046054A (en) | 2001-02-20 |
JP4402207B2 JP4402207B2 (en) | 2010-01-20 |
Family
ID=16742095
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JP21985399A Expired - Lifetime JP4402207B2 (en) | 1999-08-03 | 1999-08-03 | Alcohol-resistant yeast |
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JP (1) | JP4402207B2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005095109A (en) * | 2003-09-26 | 2005-04-14 | Kirin Brewery Co Ltd | Method for producing fermented malt beverage |
JP2009178087A (en) * | 2008-01-30 | 2009-08-13 | Kyoto Univ | Method for producing liquors or fermented seasoning |
JP2015177782A (en) * | 2014-06-18 | 2015-10-08 | 黄桜株式会社 | Method of producing liquor or fermented seasoning |
-
1999
- 1999-08-03 JP JP21985399A patent/JP4402207B2/en not_active Expired - Lifetime
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2005095109A (en) * | 2003-09-26 | 2005-04-14 | Kirin Brewery Co Ltd | Method for producing fermented malt beverage |
JP2009178087A (en) * | 2008-01-30 | 2009-08-13 | Kyoto Univ | Method for producing liquors or fermented seasoning |
JP2015177782A (en) * | 2014-06-18 | 2015-10-08 | 黄桜株式会社 | Method of producing liquor or fermented seasoning |
Also Published As
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JP4402207B2 (en) | 2010-01-20 |
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